| In 1990, Kitagawa et al first found that short ischemic episodes to the brain could induce protection against a subsequent lethal cerebral ischemic injury in gerbil model of globe cerebral ischemia. This phenomenon is known as ischemic preconditioning (IPC) and ischemic tolerance in brain. The inherent limitations of IPC limit its wide application in clinic. People had been trying to implore non-ischemic preconditioning methods, especially pharmacological preconditioning, mimicking IPC-induced ischemic tolerance, and wanted to find a practicable neuroprotective measure for the clinical application.To date, two cannabinoid (CB) receptors have been identified by molecular cloning: the CB1 and CB2 receptors. The CB1 receptor is abundantly expressed in brain tissue, but is also present in peripheral tissues including vasculature, heart and liver. The CB2 receptor was previously considered to be expressed primarily in immune and haematopoietic cells. However, more recent studies have also found CB2 receptors in brain, myocardium, cardiomyoblasts and endothelial cells of various origins. Numerous studies have demonstrated that WIN 55,212-2, a synthetic CB1/2 receptors agonist, attenuated hippocampal neuronal loss following transient global cerebral ischaemia in rats and reduced infarct size after permanent focal cerebral ischemia induced by middle cerebral artery occlusion, in a CB1-dependent manner. However, there is no report in the literature on whether preconditioning with WIN 55,212-2 could induce cerebral ischemic tolerance and alleviate cerebral ischemic injury.In the present study, using the model of focal cerebralischemia in rats induced by middle cerebral artery occlusion, we investigate that the dose-effect relationship and time-effect relationship of ischemic tolerance induced by WIN 55,212-2 preconditioning and explore the role of phosphorylated ERK1/2 (pERK1/2) in WIN 55,212-2 preconditioning.Experiment 1. Neuroprotective effect of preconditioning with different doses of WIN 55,212-2 on focal cerebral ischemia in ratsObjective: To explore the neuroprotective effect of preconditioning with different doses of cannabinoid receptor agonist WIN 55,212-2 on focal cerebral ischemia in rats .Methods: Fifty male SD rats were randomly assigned to five groups (n=10 each). The control group rats were intraperitoneally injected normal sodium 0.3ml 24h before focal cerebral ischemia; DMSO group in which dimethyl sulfoxide (DMSO) was intraperitoneally administered at the same volume as that used for preconditioning groups 24h before focal cerebral ischemia; WIN 55,212-2 preconditioning groups (WIN1~3) in which WIN 55,212-2 was intraperitoneally injected at the doses of 0.3, 1 or 3mg/kg respectively 24h before focal cerebral ischemia. Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO) for 120min. The neurological function scores (NFS) was evaluated at 24h,48h and 72h after reperfusion. The rats were then decapitated, the brain infarct volume was then assessed with 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining at 72h after reperfusion, was expressed as percentage of normal cerebral hemisphere volume.Results: (1) The neurological function scores: the NFS of rats in WIN 55,212-2 preconditioning groups was significantly higher than that in control group and DMSO group at 24h, 48h, 72h after reperfusion. (P<0.05).(2)Brain infarct volumes: the infarct volume in WIN 55,212-2 preconditioning groups was significantly smaller than that in Control group and DMSO group (P<0.05) .Moreover, the infarct volume in WIN2 and WIN3 groups was significantly smaller than that in WIN1 group. However, there was no statistical significance in the infarct volumes between WIN2 and WIN3 groups.Conclusion: Cannabinoid receptors agonist WIN 55,212-2 preconditioning protects the rat brain against transient focal cerebral ischemia injury in a dose-dependent manner.Experiment 2. Neuroprotective effect of repeated WIN 55,212-2 preconditioning on focal cerebral ischemia in ratsObjective: To explore the neuroprotective effect of repetitive cannabinoid receptor agonist WIN 55,212-2 preconditioning on focal cerebral ischemia in rats.Methods: Fifty male SD rats were randomly assigned to five groups (n=10 each). The rats in control group and DMSO group was intraperitoneally administered 0.3ml normal saline and 0.3ml DMSO once a day for 5d respectively. WIN 55,212-2 preconditioning groups (WIN1, 3, 5) in which animals was respectively given intraperitoneal injection of 1 mg kg WIN 55,212-2 (dissolved with 0.3ml DMSO) of once a day for 1 d, 3d and 5d. At 24h after last pretreatment, the focal cerebral ischemia was produced by the right middle cerebral artery occlusion (MCAO) for 120 min. The neurological function scores (NFS) was evaluated at 24h,48h and 72h after reperfusion. The brain infarct volume was then assessed with 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining at 72h after reperfusion, was expressed as percentage of normal cerebral hemisphere volume.Results: (1) The NFS of rats in WIN 55,212-2 preconditioning groups was significantly higher than that in control group and DMSO group at 24h, 48h, 72h after reperfusion (P<0.05). The infarct volume in WIN 55,212-2 preconditioning groups was significantly smaller than that in control group and DMSO group (P<0.05) (.2)The infarct volume in WIN5 groups was significantly smaller than that in WIN1 and WIN3 group. The NFS of rats in WIN5 groups was higher than that in WIN1 and WIN3 group. However, there was no statistical significance in the infarct volumes between WIN1 and WIN3 group.Conclusion: The neuroprotective effect of WIN 55,212-2 preconditioning is enhanced by increased time of pretreatment.Experiment 3. Effect of WIN 55,212-2 preconditioning on phosphorylated ERK1/2 and neuronal apoptosis following focal cerebral ischemia in ratsObjective: To study effect of WIN 55,212-2 preconditioning on phosphorylated ERK1/2 (pERK1/2) and neuronal apoptosis following focal cerebral ischemia in rats.Methods: Twenty four male SD rats were randomly assigned to four groups (n=6 each). The rats in sham group received the same the operation except without middle cerebral artery occlusion. The animals in control, DMSO and WIN 55,212-2 preconditioning groups were intraperitoneally administered 0.3ml normal saline, 0.3ml DMSO and 1mg/kg WIN 55,212-2 (dissolved with 0.3ml DMSO) once a day for 5d respectively. At 24h after last pretreatment, the focal cerebral ischemia was produced by the right middle cerebral artery occlusion (MCAO) for 120 min. All animals were decapitated at 4h after reperfusion. The pERK1/2 expression was determined by western blotting and the immunohistochemical staining, and the neuronal apoptosis was identified by TUNEL staining.Results: (1) Compared with DMSO group and control group,the numbers of apoptotic cells in WIN 55,212-2 preconditioning group were significantly lower (P<0.05); (2)The numbers of pERK1/2-like immunoreactive neurons in WIN 55,212-2 preconditioning group, DMSO group and control group were increased compared to sham group (P<0.05). The numbers of pERK1/2-like immunoreactive neurons in DMSO group and control group were significantly fewer than that in WIN 55,212-2 preconditioning group (P<0.05). There was no statistical significance in the infarct volumes between control group and DMSO group. (3) The pERK1/2 expressions in WIN 55,212-2 preconditioning group, DMSO group and Control group were increased than Sham group (P<0.05). Tthe level of pERK in WIN 55,212-2 preconditioning group was much higher than that in DMSO group and control group. There was no statistical significance in the infarct volumes between control group and DMSO group.Conclusion: Cannabinoid receptors agonist WIN 55,212-2 preconditioning up-regulates the levels of phosphorylated ERK1/2 and reduces the neuronal apoptosis.Summary1. Cannabinoid receptors agonist WIN 55,212-2 preconditioning can induce the cerebral tolerance and protect the rat brain against transient focal cerebral ischemic injury. 2. The cannabinoid receptors agonist WIN 55,212-2 preconditioning induced neuroprotective effect in a dose-dependent manner and repeated pretreatment can produce better protective effects than single preconditioning.3. Cannabinoid receptor agonist WIN 55,212-2 preconditioning can up-regulate the levels of phosphorylated ERK1/2 and reduce neuronal apoptosis, indicating the activation of phosphorylated ERK1/2 participated in the cerebral tolerance induced by cannabinoid receptor agonist WIN 55,212-2 preconditioning.
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